| Background and Objective:Polycystic ovary syndrome(PCOS)is the most common reproductive endocrine disorder that leads to infertility in women of reproductive age.PCOS individuals have multiple antral follicles but not dominant follicle maturation,and numerous follicles undergo atresia,which is the etiology of infertility.In recent years,assisted reproductive technology(ART)has developed rapidly and has become the primary method for solving infertility problems in PCOS patients.However,there are still some PCOS patients who have not achieved satisfactory clinical outcomes after ART treatment.The reason is likely to be the abnormal follicular development in PCOS patients,resulting in poor quality of oocytes and leading to low embryonic developmental potential in later stages.However,there is still a lack of effective treatment for PCOS follicular dysplasia in clinical practice,mainly because the related molecular mechanisms of PCOS are still not fully understood.Previous studies have confirmed that ovarian granulosa cells(GCs)play an essential role in the development and maturation of follicles,and the level of their proliferation and apoptosis is a crucial factor in the normal development of follicles.In recent years,it has been reported that the proliferation level of GCs is decreased in abnormal PCOS follicles,suggesting that the dysregulation of GCs proliferation ability may be related to the abnormal development of PCOS follicles.Therefore,to explore the molecular mechanism of abnormal proliferation of GCs in PCOS patients can provide some important clues for finding new diagnoses and treatment strategies.PATL2 is an m RNA-binding protein(m RNA-binding protein,m RNP),and its PAT structure can bind to target m RNA and regulate its post-transcriptional translation.The reduction or deletion of PATL2 expression significantly inhibited the development and maturation of human oocytes by regulating the spindle formation,resulting in abnormal follicular development.Surprisingly,our previous study found that attenuated PATL2 expression was also closely associated with a decreased GCs number in abnormal follicles in PCOS,and the level of granulosa cell proliferation was regulated by adrenomedullin2(ADM2).We speculate that the PAT structure of PATL2 protein may bind to the m RNA of ADM2,then protect the m RNA from degradation,and promot ADM2 translational expression,finally favoure granulosa cell proliferation.This study will investigate the molecular regulatory mechanism of granulosa cells proliferation in abnormal PCOS follicles.Our findings will help us to effectively understand the molecular mechanism of abnormal follicle development in PCOS patients,improve the quality of follicles in patients with PCOS,increase the productive capacity of their oocytes,and implement new diagnosis and treatment ideas and methods.It is essential both conceptually and in terms of possible practical application.Methods:1.According to the Rotterdamcriteria,51 PCOS patients who received ART treatment in the Reproductive Center of Maternal and Child Health Care Hospital affiliated to Nanchang University from January 2020 to December 2020 were enrolled,and 52 non-PCOS infertility patients with regular menstruation who received ART treatment in the Assisted Reproductive Center of our hospital during the same period were enrolled as the control group.The clinical data of the two groups were collected and analyzed.2.Follicular fluids were collected from the two groups,and granulosa cells were isolated in vitro.The proliferation of granulosa cells in the two groups was detected by EDU.The protein and m RNA expression levels of PATL2 and ADM2 in granulosa cells of the two groups were detected by immunofluorescence,Western Blot and RT-PCR,respectively.3.PATL2-si RNA was constructed and introduced into KGN cells to knock down the PATL2 gene.Western Blot and RT-PCR were used to detect the protein and m RNA expression levels of PATL2 and ADM2 in KGN cells,respectively.Meanwhile,CCK8 was used to detect the proliferation levels of KGN cells.4.The mutant plasmid of the "PAT" structural region of PATL2 was constructed and introduced into KGN cells to establish mutant cells.The protein and m RNA expression levels of PATL2 and ADM2 in cells were detected by Western blot and RT-PCR respectively,while the proliferation level of KGN cells was detected by CCK8.5.The rat model of PCOS was established,the proliferation of granulosa cells in ovarian tissue was detected by immunohistochemistry for Ki-67,and the protein expression levels of PATL2 and ADM2 in ovarian tissue was detected by immunohistochemistry.The protein expression levels of PATL2 and ADM2 in ovarian tissues were detected by Western blot;The gene expression levels of PATL2 and ADM2 in ovarian tissues was detected by RT-PCR.Results:(1)According to the inclusion,exclusion criteria and infertility criteria,a total of103 patients were enrolled in this study,including PCOS group(51 patinets)and control group(52 patients).The clinical data of the two groups showed that there were no significant differences in age,FSH,E2 and PRL between the PCOS group and the control group(P>0.05),and the body weight,LH and T of the PCOS group were significantly higher than those of the control group(P<0.05).After Gn treatment,the oocyte maturation rate and fertilization rate in the PCOS group were significantly lower than those in the control group,and there were significant differences(P<0.05).However,there were no significant differences in the cleavage rate and optimal embryo rate between the two groups(P>0.05).(2)The EDU results showed that the proliferation levels of granulosa cells in the PCOS group was significantly lower than that in the control group,and the difference was statistically significant(P<0.05).Immunofluorescence results showed that PATL2 protein was mainly expressed in the cytoplasm of granulosa cells of follicles.Compared with the control group,the expression levels of PATL2 in the granulosa cells of the PCOS group was significantly reduced;Western Blot and RT-PCR results showed that compared with the control group,the expression levels of PATL2 and ADM2 in granulosa cells in the PCOS group were significantly decreased(P<0.05).(3)After knocking down PATL2 by si RNA in KGN cells,the expression levels of ADM2 was significantly reduced.Furthermore,the proliferation ability of KGN cells in the PATL2 knockdown group was also significantly attenuated.(4)The results of CCK8 detection showed that the MT group with deletion of the "PAT" region had significantly attenuated cell proliferation ability compared with the WT group with the typical "PAT" region.Western Blot and RT-PCR showed that although there was no difference of PATL2 expression between these two groups,the AMD2 expression levels in MT group was significantly decreased when compared to WT group.(5)Animal experiments showed that the proliferation levels of the granulosa cells in PCOS group was remarkably lower than that in control group.Moreover,the expression levels of PATL2 and ADM2 in the PCOS model group were obviously decreased.Conclusions:(1)The follicular development of PCOS patients was abnormal,and the proliferation levels of granulosa cells was significantly weakened,suggesting that the attenuated granulosa cell proliferation ability is closely related to the abnormal follicular development of PCOS.(2)In vitro cell experiments,PATL2 promoted the proliferation of granulosa cells by regulating the expression of ADM2,suggesting that PATL2 deficiency may inhibit granulosa cells proliferation by down-regulating the expression of ADM2,which may be one of the critical reasons resulting in the abnormal development of PCOS follicles.(3)In vivo animal expertments showed that PATL2 and ADM2 expression levels were significantly reduced in granulosa cells in dysfunctional PCOS follicles,and the granulosa cell’s proliferation capacity was significantly reduced. |
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